Long noncoding RNA SNHG1 promotes breast cancer progression by regulating the miR-641/RRS1 axis

An increasing number of studies have indicated the crucial involvement of long non-coding RNAs (lncRNAs) in the onset and progression of malignancies. However, a complete understanding of the molecular mechanism underlying the effect of abnormally expressed lncRNAs on breast cancer (BC) remains elusive. This study aimed to elucidate the influence of the lncRNA small nucleolar RNA host gene 1 (SNHG1) on BC progression and its underlying mechanism. Our findings revealed a conspicuous up-regulation of SNHG1 in both BC tissues and cells. The downregulation of SNHG1 was observed to inhibit BC cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) processes, while simultaneously promoting apoptosis. Furthermore, dual-luciferase reporter gene and RNA pull-down assays established that SNHG1 targeted miR-641 expression, while miR-641 targeted RRS1. Rescue studies demonstrated that in vitro SNHG1 silencing could be reversed by the miR-641 inhibitor, as well as by RRS1 upregulation. Moreover, in vivo downregulation of SNHG1 was found to inhibit BC growth. Through the inhibition of the miR-641 level, SNHG1 elevated the level of the downstream target RRS1, thereby fostering BC growth, migration, and invasion while inhibiting apoptosis. These findings suggest that SNHG1 may represent a potential therapeutic target for BC treatment.


Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was initially extracted from BC cells and tissues using the TRIzol reagent (Invitrogen, Waltham, MA, USA), and RNA quantity was determined by spectrophotometry.The HiScript III All-in-one RT SuperMix kit (Vazyme, Nanjing, China) was utilized to convert RNA to cDNA.For miRNA, the miRNA first strand cDNA synthesis kit (Vazyme) employed the stem-loop method.qRT-PCR was performed using the ChamQ Universal SYBR qPCR Master Mix (Vazyme).The relative abundance of RNA genes was calculated using the 2 −ΔΔ Ct method.The list of primers used is provided below.

Western blotting
Cells, washed twice with phosphate-buffered saline (PBS), were fully lysed using RIPA, and protein quantification was performed using the BCA kit (Shanghai Epizyme Biomedical Technology, China).Protein samples were added to a quarter-volume 5× sodium dodecyl sulfate buffer solution, boiled for 10 min, separated by SDS-PAGE, and then transferred to a PVDF membrane (Because the molecular weights of the target proteins are very close, the length of the trimmed PVDF membrane is relatively short).After blocking the membranes with 5% skim milk and Tris-buffered saline in Tween-20 at 25 °C, they were incubated overnight at 4 °C with anti-GAPDH (1:1000), anti-N-cadherin (1:1000), anti-E-cadherin (1:1000), and anti-RRS1 (1:1000) antibodies (all purchased from Abcam, Cambridge, UK).Membranes were washed with TBS-T and then incubated with a horseradish peroxidase-labeled secondary antibody (1:1000; Abclonal, Woburn, MA, USA) for 1 h.Membranes were visualized with an enhanced chemiluminescence kit, and protein expression was measured using Image J v1.53t.GAPDH served as the internal control.

Dual-luciferase reporter gene assay
The wild-type (WT) and corresponding mutated (MUT) sequences of SNHG1 and RRS1, binding to miR-641, were synthesized and cloned into the pSI-check2 (Hanbio Biotechnology, Shanghai, China) reporter vector.MCF-7 and BT-549 cells were cultured in 24-well plates, and reporter vectors were co-transformed with miR-641 mimics or NC using LipoFiter 3.0™ (Hanbio Biotechnology, Shanghai, China).After 48 h of cell culture, relative luciferase activity was measured using the dual-luciferase reporter assay system (Promega, Madison, WI, USA).

Colony formation assay
Transfected cells were plated in 6-well plates at a density of 5 × 10 2 cells per well and cultured in 10% FBS medium for 14 days.Colonies were fixed with 4% paraformaldehyde at 25 °C and stained with 600 mL of 5% crystal violet in each well for 20 min.Manual counting of colonies was performed after staining.

Cell counting kit 8 (CCK-8) assay
To assess the impact of SNHG1 on BC cells, 3 × 10 3 cells were seeded in 96-well plates with five replicates per group.At 0, 1, 2, 3, and 4 days of culture, 10 μL of the CCK-8 kit (Solarbio, Beijing, China) was added to each well to detect cell proliferation and incubated at 37 °C for 120 min.Absorbance at 450 nm was measured using a microplate reader.

Flow cytometry assay
Cells, digested with 0.25% trypsin and washed twice with PBS, were collected (1 × 10 6 cells).Subsequently, 2.5 μL Annexin V-APC and 2.5 μL PI staining fluid were added to the cell suspension and incubated for 15 min.The rate of apoptosis was assessed using the CytoFLEX LX flow cytometer (Beckman Coulter, Brea, CA, USA).

Transwell assays
To evaluate the impact of SNHG1 on cell invasion and migration, transwell chambers coated with Matrigel (Corning, NY, USA) or uncoated were employed.Transfected cells (2 × 10 4 ) were added to the upper chamber in 200 μL serum-free medium, while 600 μL 20% serum medium was placed in the bottom chamber.After 24 h cell culture, cells that migrated or invaded the membrane were fixed with methanol and stained with 0.5% crystal violet at 25 °C.Subsequently, cells were observed and counted under a microscope.

RNA pulldown assays
BC cells were transfected with 100 nM biotin-labeled miR-641 or negative control probes (GenePharma, Shanghai, China).After 48 h cultivation, 1 × 10 7 cells were harvested and lysed.RNA pull-down experiments were conducted using the RNA Pull-Down Kit (BersinBio, Guangzhou, China).Streptavidin magnetic beads and cell lysates were mixed and incubated overnight at 4 °C with rotation, following the manufacturer's instructions.Subsequently, captured SNHG1 was purified with TRIzol and analyzed using qRT-PCR.

In vivo experiments
Animal experiments were conducted with approval from the Qingdao University Laboratory Animal Welfare Ethics Committee (approval number: 202207BALB/cnude12202208082, date: 5 June 2022).All procedures adhered to relevant guidelines and regulations and were reported in accordance with ARRIVE guidelines.BALB/c nude mice (4 weeks old) were supplied by Beijing Vital River Laboratory Animal Technology (China).Sh-SNHG1 or negative control MCF-7 cells (5 × 10 6 ) were resuspended in a 200 μL mixture of 100 μL PBS with 100 μL Matrigel (Corning) and subcutaneously injected into the anterior part of mice (n = 5 per group).Tumor volume was calculated using the formula V = (length × width 2 )/2 every 3 days after tumor formation.On day 24, mice were euthanized using the cervical dislocation method under anesthesia (0.7% sodium pentobarbital), and tumors were harvested and weighed.Tumor tissues were fixed with paraformaldehyde, embedded in paraffin, and cut into 5-μm sections for immunohistochemistry (IHC).The primary antibody RRS1 (1:200, ab188161; www.nature.com/scientificreports/Abcam) was used for IHC, with nuclei stained blue with hematoxylin and RRS1-positive level appearing brown or brownish yellow with diaminobenzidine (Supplementary Information).

Statistical analysis
All experimental data were analyzed using SPSS v13.0 software and GraphPad Prism v8.0 software.The data were presented as mean ± standard deviation (SD), and differences between groups were determined by Student's t-test.P-value < 0.05 was considered statistically significant.

SNHG1 expression is elevated in BC
Upon analysis of the The Cancer Genome Atlas (TCGA) public database, it was observed that SNHG1 expression was significantly elevated in BC compared to normal tissues (Fig. 1A).Using qRT-PCR, we further investigated SNHG1 expression in BC tissues and found it to be notably higher than in paired normal tissues (Fig. 1B).

Knockdown of SNHG1 inhibited BC cell proliferation, migration, invasion, and induced apoptosis
To investigate the impact of SNHG1 on BC cells in vitro, we selected BT-549 and MCF-7, which exhibited relatively high SNHG1 expression, and used siRNA to interfere with SNHG1 expression.qRT-PCR analysis revealed that SNHG1 expression decreased in cells transfected with three types of SNHG1 siRNA compared to the control group, with the most effective knockdown observed with si-SNHG1-1 (Fig. 2A).Consequently, si-SNHG1-1 was employed in subsequent research.The CCK8 assay and colony formation assay indicated that SNHG1 knockdown significantly inhibited BC cell proliferation and reduced clone formation (Fig. 2B,C).Additionally, transwell assays were used to assess the effects of SNHG1 on BC cell migration and invasion, revealing a marked decrease in both abilities following SNHG1 knockdown (Fig. 2D,E).EMT, closely associated with tumor invasion and migration, was analyzed by Western blot.The level of EMT-related protein N-cadherin significantly decreased after SNHG1 silencing, while the level of E-cadherin increased (Fig. 2F).Flow cytometry analysis demonstrated that SNHG1 knockdown induced BC cell apoptosis (Fig. 2G).These findings indicate that the lncRNA SNHG1 plays a crucial role in regulating BC proliferation, migration, invasion, and apoptosis.

SNHG1 regulates the expression of miR-641
An increasing number of studies have indicated that lncRNAs can bind to miRNAs, acting as competing endogenous RNAs (ceRNAs) 21 .The level of miR-641 in BC tissues and cells was assessed using qRT-PCR, revealing downregulation in BC tissues and cells (Fig. 3A,B).In BT-549 and MCF-7 cells, silencing SNHG1 expression led to an increase in the level of miR-641 (Fig. 3C).The sequence complementarity between SNHG1 and miR-641 was predicted using Starbase v2.0 (http:// starb ase.sysu.edu.cn/) (Fig. 3D).Subsequently, the dual-luciferase reporter gene assay was conducted to validate the binding relationship between SNHG1 and miR-641.MiR-641 mimics significantly reduced the luciferase activity of SNHG1-WT, but did not affect the luciferase activity of SNHG1-MUT (Fig. 3E,F).Additionally, RNA pull-down experiments demonstrated a significant increase in the ratio of endogenous SNHG1 pulled down by biotin-labeled miR-641 (Fig. 3G).In conclusion, our findings demonstrate that SNHG1 can directly bind to miR-641, suggesting that the oncogenic mechanism of SNHG1 may involve the negative regulation of miR-641.

RRS1 is directly targeted by miR-641 and upregulated in BC
To further investigate the mechanism of SNHG1 action in BC, we utilized the targetScan database (https:// www.targe tscan.org/ vert_ 72/) to identify miR-641's downstream target gene RRS1.Analysis using the GEPIA database (http:// gepia.cancer-pku.cn/), revealed elevated RRS1 mRNA levels in BC tumor tissues compared to normal tissues in the TCGA cohort (Fig. 4A).Similarly, Western blot results demonstrated a significant increase in RRS1 protein level in cancer tissues compared to BC-paired normal tissues (Fig. 4B).Moreover, RRS1 was found to be upregulated in BC cell lines (Fig. 4C).Subsequent dual-luciferase reporter gene assays confirmed the binding of miR-641 to the 3'-UTR of RRS1 as predicted by TargetScan (Fig. 4D,E).MiR-641 upregulation markedly inhibited RRS1 protein level, while miR-641 downregulation increased RRS1 protein level (Fig. 4F).We further explored the regulation of RRS1 level by SNHG1.After silencing SNHG1, the protein level of RRS1 significantly decreased (Fig. 4G).These results indicate that RRS1 is a direct target of miR-641 and is regulated by SNHG1.

Down-regulation of miR-641 or upregulation of RRS1 reverses the effect of SNHG1 silencing on BC cell
To explore whether SNHG1 mediates the impact of miR-641 and RRS1 on BC growth, migration, invasion, and apoptosis, rescue experiments were conducted by down-regulating miR-641 or upregulated RRS1 in the context of SNHG1 silencing.Silencing SNHG1 inhibited the growth, migration, and invasion, as well as promoted apoptosis of BC cells, but these effects were partially reversed by down-regulation of miR-641 or restoration of RRS1 level (Fig. 5A-E).Additionally, transfection with the miR-641 inhibitor or upregulation of RRS1 also counteracted the changes in EMT-related proteins and RRS1 level observed after silencing SNHG1 (Fig. 5F).Overall, these findings further underscore that SNHG1 promotes the progression of BC cells through the miR-641/RRS1 axis.

SNHG1 intervention inhibits the growth of BC tumors in vivo
To delve into the role of SNHG1 in tumorigenesis in vivo, SNHG1-stable-knockdown MCF-7 cells were subcutaneously injected into nude mice to establish a xenograft mouse model.It was observed that tumor size, weight, and growth curves were significantly suppressed after SNHG1 silencing (Fig. 6A-C).The xenograft tumors derived from the sh-NC group exhibited higher SNHG1 levels (Fig. 6D).Additionally, IHC results demonstrated a decrease in RRS1-positive cells in tumor tissues following SNHG1 knockdown (Fig. 6E).The downregulation of SNHG1 can effectively inhibit the growth of BC tumors in vivo.

Discussion
BC is a highly heterogeneous disease, and the overall treatment effect is not ideal.Therefore, exploring new treatment methods to reduce BC mortality remains the focus of public attention 22,23 .The dysregulation of lncRNA is strongly associated with the development of various malignant tumors, including BC 24 .Consequently, an increasing number of studies are concentrating on investigating the functions and regulation of lncRNAs to identify new targets for cancer diagnosis and treatment.SNHG1 comprises 8 small nucleolar RNAs with 11 exons 14 .Currently, there are limited studies on the function and mechanism of SNHG1 in BC.Previous research has indicated that SNHG1 is highly expressed in hepatocellular carcinoma, where it mediates sorafenib resistance through activating the AKT pathway and is linked to poor prognosis 25 .Similarly, our experiments revealed up-regulation of SNHG1 in BC tissues and cells.Moreover, accumulating evidence suggests that lncRNAs play a crucial role in regulating various biological processes in BC, including proliferation and metastasis.For instance, Xue et al. demonstrated that lncRNA AC073352.1 binds to YBX1 to promote BC metastasis and angiogenesis 8 .In prostate cancer, SNHG1 promotes EMT processes by competitively binding to hnRNPL, preventing E-cadherin translation 16 .Our in vivo results indicated that the downregulation of SNHG1 suppressed BC progression.Consistent with our findings, interference with lncRNA Multiple studies have unveiled that lncRNA, operating as a ceRNA by sequestering miRNAs, plays a crucial role in regulating the biological functions of tumors 27,28 .Previous investigations have indicated the presence of SNHG1 in both the cytoplasm and nucleus of BC cells 29 .According to recent research, MCM3AP-AS1 acts as a ceRNA in hepatocellular carcinoma, functioning as a sponge for miR-194 to enhance FOXA1 expression 30 .Another study has identified SNHG1 as a ceRNA that promotes gastric cancer cell proliferation by modulating the miR-140/ADAM10 axis 31 .Mechanistically, we hypothesized that SNHG1 operates as a ceRNA through miRNA sequestration.Initially, bioinformatics analysis revealed a potential binding sequence between SNHG1 and miR-641.Subsequent dual-luciferase reporter gene assays and RNA pull-down assays confirmed that SNHG1 targets miR-641.Liang et al. previously discovered that miR-641, down-regulated in glioma, acts as a sponge for COX10-AS1 19 .Moreover, studies have indicated the regulatory role of miR-641 in the BC process 32 .Rescue experiments demonstrated that a miR-641 inhibitor partially reverses the inhibitory effects of SNHG knockdown on BC proliferation, migration, invasion, and EMT processes.
We comprehensively investigated the mechanism by which miR-641 regulates RRS1.Our data demonstrated that miR-641 can modulate the level of downstream RRS1, as confirmed by the dual-luciferase reporter gene assay.RRS1 functions as an oncogene in cancer, with RRS1 gene mutation and abnormal expression promoting tumor growth and metastasis 33 .Silencing RRS1 inhibits EMT, migration, and invasion in BC cells, and RRS1 affects BC progression via the RPL11-c-Myc-SNAIL axis 20 .Down-regulation of SBF2-AS1 and up-regulation of miR-143 can inhibit proliferation and promote apoptosis in BC cells by suppressing RRS1 level 26 .The TCGA database and analysis of four pairs of BC tumor tissues revealed an elevated RRS1 level.In this study, we discovered that SNHG1 regulates BC cell proliferation, migration, invasion, EMT, and apoptosis processes.SNHG1 modulates  RRS1 in BC cells by competitively binding to miR-641, and the overexpression of RRS1 can restore the cellular functions mediated by SNHG1.Moreover, ribosome biosynthesis is one of the major functions of RRS1, and whether SNHG1 can mediate BC ribosome biosynthesis and its downstream molecular mechanisms through RRS1 needs to be thoroughly investigated.

Conclusion
This study marks the first investigation into the molecular mechanism involving SNHG1, miR-641, and RRS1.
Our findings confirmed the binding interaction between SNHG1 and miR-641, establishing that the silencing of SNHG1 could inhibit EMT, proliferation, migration, and invasion while promoting apoptosis in BC cells.This effect was achieved through the inhibition of the miR-641/RRS1 axis (Fig. 7).Consequently, our study provides new insights for molecularly targeted therapy in BC.

Figure 1 .Figure 2 .
Figure 1.SNHG1 is overexpressed in breast cancer (BC).(A) Comparison of SNHG1 expression in human BC tissues based on the TCGA data set.(B) Expression of SNHG1 in 21 paired BC tissues and normal tissues.(C) SNHG1 expression in BC cell lines and the MCF-10A cell line.***P < 0.001.

Figure 4 .
Figure 4. RRS1 is overexpressed in BC and targeted by miR-641.(A) GEPIA confirms the obvious overexpression of RRS1 in BC tissues based on the TCGA cohort.(B) The level of RRS1 in four pairs of BC tissues was analyzed by Western blot.(C) Protein levels of RRS1 in BC cells.(D) Binding sites of RRS1 and miR-641.(E) MiR-641 specifically targets RRS1.(F) Relative level of RRS1 protein after miR-641 overexpression or knockdown.(G) Relative level of RRS1 after knockdown of SNHG1.*P < 0.05; **P < 0.01.

Figure 5 .
Figure 5. Altering the expression of miR-641 and RRS1 reversed the effect of silencing SNHG1.(A, B) Cell growth was measured using the CCK-8 assay (A) and the colony formation assay (B).(C, D) Transwell assay was used to detect cell migration (C) and invasion (D).(E) Detection of cell apoptosis by flow cytometry.(F) The levels of RRS1 and EMT-related proteins detected by western blotting.**P < 0.05; ***P < 0.001.